Low sample volume instruments that work in the range of 2 microliters or less are particularly useful in the quantitation of biotechnology samples including nucleic acids, proteins and drugs and any other liquid samples where it is desirable to keep the volume of the sample loss to a minimum where available analyte quantity is very limited and where convenience of measurement is desired.
Prior art concentrates on the containment of samples in vessels or containers wherein the sample volume is usually from 20 to 1000 microliters. A relatively straightforward spectrofluorometer design can be seen in Nogami et al. U.S. Pat. No. 5,500,536. A spectrofluorometer utilizing simultaneous multiple source wavelengths can be seen in Goldstein U.S. Pat. No. 5,422,719. Use of optical fibers in the spectrofluorometer optical system can be seen in Glebeler et al. U.S. Pat. No. 6,313,471. A good general introduction to fluorescence can be found in “Principles of Fluorescence Spectroscopy” by Joseph R. Lakowitz, 1999, Kluwer Academic/Plenum Publishers, 233 Spring Street, New York, N.Y., 10013, pages 1 to 9.
Robertson, in U.S. Pat. Nos. 6,628,382 and 6,809,826 discloses method and apparatus for photometric or spectrophotometric measurements on extremely small samples. These “nanodrop” samples, as they are termed therein and herein, are on the order of 2 microliters or less and are contained by surface tension. These patents are incorporated in their entirety by reference. In the apparatus of Robertson, a nanodrop is contained by surface tension between two relatively moveable, substantially parallel surfaces, “anvils”, which are moved together after the sample is loaded upon one of them in order to wet both surfaces. The anvils are then moved apart to draw the droplet into a column to establish an optical path through the length of which light is projected. In-line optics are used to pass light through the column. The light passes from an input optical fiber in one anvil to an aligned output optical fiber in the other and to a sensor, a charge coupled device or the like, which can be part of a spectrometer or other optical detection system, where a photometric or spectrophotometric measurement is made.
In U.S. Pat. No. 6,809,826, Robertson discloses an improved version of the above invention in which the wetted surface area on the anvils is limited by various means.
In these two patents Robertson teaches that fluorescence may be measured with the apparatus disclosed therein.
Measurements of the type disclosed in the referenced Robertson patents, however, are not optimally applicable to measurement of a fluorescing nanodrop. Containing the sample by surface tension is appropriate and highly effective. However the light handling system of the two, prior-art, Robertson inventions tends to overwhelm a fluoroscopic measurement particularly so when a weakly emitting or fluorescing sample is involved. Light used to excite the fluorescence projected from one in-line optical fiber through the drawn column of fluid to excite fluorescence in the contained nanodrop and directly into an in-line receiving optical fiber to a sensor interferes with the much less intense light produced by the sample fluorescence itself. In addition, fluorescence from the optical fibers would be high for some exciting wavelengths.
It is therefore an object of this invention to provide method and apparatus for performing fluorescence measurements on nanodrops contained by surface tension wherein transmittance of emitted fluorescent light received by the sensing system is maximized and light from other sources, ambient illumination, fluorescence from the system optical fibers and particularly scattered light from the exciting source, that is received by the sensing system is minimized.
It is a further object of this invention to provide method and apparatus for the measurement of fluorescence wherein a nanodrop sample is contained by surface tension and the exciting light, and any stray ambient light is substantially barred from the measuring detection system.
It is a still further object of this invention to provide method and apparatus for the measurement of fluorescence emitted by a nanodrop sample contained by surface tension wherein compensation is substantially provided for any exciting and ambient light that does impinge on the measuring sensor.